During metanephric the epithelial cells in different segments of the nephron and collecting duct begin to express protein markers characteristic of the mature, differentiated cell. We have established in prior work that amplified expression of the vacuolar H+ATPase, containing the B1 isoform of the B subunit, is a marker for intercalated cell differentiation, and is first detectable by antibody staining on gestational day 17. In studies using transgenic mice and cultured cells, we have established a region of the B1 subunit gene that confers intercalated cell-specific expression. We have recently discovered that a GAn ("GAGA") box in this region is essential for expression of the B1 isoform. The long term goals of this application are to understand the mechanisms controlling renal epithelial cell differentiation. The specific aims of this proposal are: l. To define the time course for expression of mRNA for vacuolar H+ATPase subunits during embryonic kidney development in vivo using in situ hybridization. 2. To isolate and characterize the GAn-binding protein(s) required for B1 subunit isoform expression. Oligonucleotide screening of a kidney bacterial expression library, or the one-hybrid method for screening a yeast GAL4 activation domain plasmid library, will be used to isolate clones for the GAn-binding proteins. The DNA binding properties and expression of the proteins in kidney will then be examined. 3. To define the promoter elements on the kidney isoform of the kidney vacuolar H+ATPase required for amplification of expression during development. Promoter-reporter constructs will be assayed in cultured cells, kidney tissue, and intact mice using several approaches, to identify cis-regulatory elements essential for B1 subunit expression. These studies should advance our understanding of developmental abnormalities of the collecting duct, and may provide information pertinent to mechanisms controlling renal epithelial cell differentiation that occurs following ischemic renal injury.